MODIFIED RNAi AGENTS

ABSTRACT

One aspect of the present invention relates to double-stranded RNAi (dsRNA) duplex agent capable of inhibiting the expression of a target gene in vivo. The dsRNA duplex comprises one or more xylo modifications in one or both strand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for in vivo therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

FIELD OF THE INVENTION

The invention relates RNAi duplex agents having particular motifs thatare advantageous for inhibition of target gene expression in vivo, aswell as RNAi compositions suitable for in vivo therapeutic use.Additionally, the invention provides methods of inhibiting theexpression of a target gene by administering these RNAi duplex agents,e.g., for the treatment of various diseases.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNAi (dsRNA)can block gene expression (Fire et al. (1998) Nature 391, 806-811;Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directsgene-specific, post-transcriptional silencing in many organisms,including vertebrates, and has provided a new tool for studying genefunction. RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.

Double-stranded RNA (dsRNA) molecules with good gene-silencingproperties are needed for drug development based on RNA interference(RNAi). An initial step in RNAi is the activation of the RNA inducedsilencing complex (RISC), which requires degradation of the sense strandof the dsRNA duplex. Sense strand was known to act as the first RISCsubstrate that is cleaved by Argonaute 2 in the middle of the duplexregion. Immediately after the cleaved 5′-end and 3′-end fragments of thesense strand are removed from the endonuclease Ago2, the RISC becomesactivated by the antisense strand (Rand et al. (2005) Cell 123, 621).

It was believed that when the cleavage of the sense strand is inhibited,the endonucleolytic cleavage of target mRNA is impaired (Leuschner etal. (2006) EMBO Rep., 7, 314; Rand et al. (2005) Cell 123, 621; Schwarzet al. (2004) Curr. Biol. 14, 787). Leuschner et al. showed thatincorporation of a 2′-O-Me ribose to the Ago2 cleavage site in the sensestrand inhibits RNAi in HeLa cells (Leuschner et al. (2006) EMBO Rep.,7, 314). A similar effect was observed with phosphorothioatemodifications, showing that cleavage of the sense strand is absolutelyrequired for efficient RNAi also in mammals.

Chemical modifications offer “drug-like” properties and modulatetherapeutic characteristics such as biostability, immune stimulation andpharmacology of short interfering RNAs (siRNA). The acceptance of extentof chemical modification on sense (or passenger) and antisense (orguide) strands are determined by the nature of the modification andpositional placement of the chemical modification in the oligonucleotidesequence in each strand. There is thus an ongoing need for iRNA duplexagents to improve the gene silencing efficacy of siRNA genetherapeutics. This invention is directed to that need.

SUMMARY

Disclosed herein are oligonucleotides comprising xylo-sugar modified or3′-modified containing for RNA interference. The modifications arepresent on the sense strand on the antisense strand or on both the sensestrand and the antisense strand thereby imparting on the moleculesbeneficial properties including one or more of increased knock downactivity of target gene expression, increased stability to endo- and orexonucleases (i.e. act as a capping moiety), reduced off-target effectsand/or lack of immunomodulating effects and are useful in the treatmentof subjects suffering from diseases or conditions and or symptomsassociated with such diseases or conditions or at risk of contractingdiseases or conditions in which target gene expression has adverseconsequences. In particular, Xylo-F and 3′-O-methyl xylosugar (Xylo-OMe)modification on siRNA stability and gene silencing activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is LC-MS of representative Xylo-F containing oligonucleotide.

FIG. 2 is a graph of IC50 values of Xylo-F. Xylo-OMe and 3′-OMe sugarmodified siRNAs to the control siRNA.

FIG. 3 is a graph showing Xylo-sugar modified siRNA duplexes were potentin vivo.

FIG. 4 is a graph showing the stability of Xylo-sugar modificationagainst exonucleases snake venon phosphodiesterases (SCPD) assay.

DETAILED DESCRIPTION

In one embodiment, one or more of xylo-sugar modifications isincorporated into a sense strand and/or antisense strand of a dsRNAagent. The dsRNA agent optionally conjugates with a GalNAc derivativeligand, for instance on the sense strand. The resulting dsRNA agentspresent superior gene silencing activity, or maintain the silencingactivity but provide stability to endo- and or exonucleases, reducedoff-target effects and/or lack of immunomodulating effects.

In one embodiment, the invention provides a double-stranded RNAi (dsRNA)agent comprising one or more xylo-modified or 3′-modified nucleosidecapable of inhibiting the expression of a target gene in vivo. The dsRNAagent comprises a sense strand and an antisense strand. Each strand ofthe dsRNA agent can range from 12-30 nucleotides in length. For example,each strand can be between 14-30 nucleotides in length, 17-30nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides inlength, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides inlength, 19-21 nucleotides in length, 21-25 nucleotides in length, or21-23 nucleotides in length. The sense strand and antisense strandtypically form a duplex dsRNA. The duplex region of a dsRNA agent may be12-30 nucleotide pairs in length. For example, the duplex region can bebetween 14-30 nucleotide pairs in length, 17-30 nucleotide pairs inlength, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs inlength, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs inlength, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs inlength, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs inlength, or 21-23 nucleotide pairs in length. In another example, theduplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, and 27. The dsRNA agent may contain one or more overhang regionsand/or capping groups of dsRNA agent at 3′-end, or 5′-end or both endsof a strand. The overhang can be 1-6 nucleotides in length, for instance2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides inlength, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides inlength. The overhangs can be the result of one strand being longer thanthe other, or the result of two strands of the same length beingstaggered. The overhang can form a mismatch with the target mRNA or itcan be complementary to the gene sequences being targeted or can beother sequence. The first and second strands can also be joined, e.g.,by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the xylo-modified nucleoside is represented byFormula A,

-   -   Y is H, alkyl or internucleotide linkage    -   X is halogen, substituted or unsubstituted alkoxy, substituted        or unsubstituted aminoalkyl, substituted or unsubstituted alkyl    -   B is a natural or non-natural nucleobase.

In one embodiment, X is F or OMe.

In one embodiment, the xylo-modified nucleoside is within the 2, 3, 4, 5or 6 of termini nucleotides at the 3′ and/or 5′ of the sense and/orantisense.

In one embodiment, the xylo-modified nucleoside is within the 2, 3, 4, 5or 6 of termini of the duplex region of the dsRNA agent at the 3′ and/or5′ of the sense and/or antisense.

In one embodiment, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 xylo-modified nucleic acid moieties at the 3′ terminal orpenultimate positions of the sense strand. In one embodiment the sensestrand comprises threose nucleic acid moieties in positions 18-19,17-18, 16-17, or 15-16 from the 5′ terminus. In one embodiment the sensestrand comprises xylo-modified nucleic acid moieties in positions 15-17,15-18 or 15-19 from the 5′ terminus. In one embodiment the sense strandcomprises xylo-modified nucleic acid moieties in positions 11-19, 12-19,13-19, 14-19, 15-19, 16-19, 17-19, from the 5′ terminus. In oneembodiment the sense strand comprises xylo-modified nucleic acidmoieties in positions 11-18, 12-18, 13-18, 14-18, 15-18, 16-18 from the5′ terminus.

In one embodiment, the sense strand comprises at least one xylo-modifiednucleic acid moieties within 1, 2, 3 or 4 position at the 3′ terminal orpenultimate positions of the sense strand, antisense strand, or bothstrands.

In one embodiment, the sense strand comprises at least one or twoxylo-modified nucleic acid moieties within 2 and/or 3 position at the 3′terminal or penultimate positions of the sense strand, antisense strand,or both strands.

In one embodiment, the sense strand comprises at least one xylo-modifiednucleic acid moieties within 1, 2, 3 or 4 position at the 5′ terminal orpenultimate positions of the sense strand, antisense strand, or bothstrands.

In one embodiment, the sense strand comprises at least one or twoxylo-modified nucleic acid moieties within 2 and/or 3 position at the 5′terminal or penultimate positions of the sense strand, antisense strand,or both strands.

In one embodiment, when the sense strand comprises at least onexylo-modified nucleic acid moieties within the internal position of theduplex, the corresponding antisense strand comprises the correspondingxylo-modified nucleic acid moieties such that the modified nucleosidesfrom the sense strands and the antisense strand are complementary toeach other. For example, the xylo-modified nucleoside in the sensestrand can be base paired with the xylo-modified nucleoside of theantisense strand.

In one embodiment, when the sense strand comprises at least onexylo-modified nucleic acid moieties within the internal position of theduplex, the corresponding antisense strand comprises the correspondingxylo-modified nucleic acid moieties such that the modified nucleosidesfrom the sense strands and the antisense strand are not base paired toeach other.

The above embodiments for xylo-modifications on nucleic acid moietiescan be used for either single-stranded or double-stranded iRNA agent.

In one embodiment, the xylo-modified nucleoside can be used in asingle-stranded RNAi agent, miRNA, or miRNA mimetic. In one embodiment,the single-stranded RNAi agent, miRNA, or miRNA mimetic agent comprisesat least one xylo-modified nucleic acid moieties within 1, 2, 3 or 4position at the 3′ terminal or penultimate positions of the strand. Inone embodiment, the single-stranded RNAi agent, miRNA, or miRNA mimeticcomprises at least one or two xylo-modified nucleic acid moieties within2 and/or 3 position at the 3′ terminal or penultimate positions of thestrand. In one embodiment, the single-stranded RNAi agent, miRNA, ormiRNA mimetic comprises at least one xylo-modified nucleic acid moietieswithin 1, 2, 3 or 4 position at the 5′ terminal or penultimate positionsof the strand. In one embodiment, the single-stranded RNAi agent, miRNA,or miRNA mimetic agent comprises at least one or two xylo-modifiednucleic acid moieties within 2 and/or 3 position at the 5′ terminal orpenultimate positions of the strand.

In one embodiment, the 3′-modified nucleoside is represented by Formula(B),

-   -   Y is H, alkyl or internucleotide linkage    -   X is halogen, substituted or unsubstituted alkoxy, substituted        or unsubstituted aminoalkyl, substituted or unsubstituted alkyl    -   B is a natural or non-natural nucleobase.        In one embodiment, X is F or OMe.

In one embodiment, the 3′-modified nucleoside is within the 2, 3, 4, 5or 6 of termini nucleotides at the 3′ and/or 5′ of the sense and/orantisense.

In one embodiment, the 3′-modified nucleoside is within the 2, 3, 4, 5or 6 of termini of the duplex region of the dsRNA agent at the 3′ and/or5′ of the sense and/or antisense.

In one embodiment the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 3′-modified nucleic acid moieties at the 3′ terminal orpenultimate positions of the sense strand. In one embodiment the sensestrand comprises threose nucleic acid moieties in positions 18-19,17-18, 16-17, or 15-16 from the 5′ terminus. In one embodiment the sensestrand comprises 3′-modified nucleic acid moieties in positions 15-17,15-18 or 15-19 from the 5′ terminus. In one embodiment the sense strandcomprises 3′-modified nucleic acid moieties in positions 11-19, 12-19,13-19, 14-19, 15-19, 16-19, 17-19, from the 5′ terminus. In oneembodiment the sense strand comprises 3′-modified nucleic acid moietiesin positions 11-18, 12-18, 13-18, 14-18, 15-18, 16-18 from the 5′terminus.

In one embodiment, the sugar modified reagent is selected from the groupconsisting of:

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 19 nt in length, wherein the sense strand contains at leastone motif of three 2′-F modifications on three consecutive nucleotidesat positions 7, 8, 9 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 20 nt in length, wherein the sense strand contains at leastone motif of three 2′-F modifications on three consecutive nucleotidesat positions 8, 9, 10 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 21 nt in length, wherein the sense strand contains at leastone motif of three 2′-F modifications on three consecutive nucleotidesat positions 9, 10, 11 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention comprises a 21nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense,wherein the sense strand contains at least one motif of three 2′-Fmodifications on three consecutive nucleotides at positions 9, 10, 11from the 5′ end; the antisense strand contains at least one motif ofthree 2′-O-methyl modifications on three consecutive nucleotides atpositions 11, 12, 13 from the 5′ end, wherein one end of the dsRNA isblunt, while the other end is comprises a 2 nt overhang. Preferably, the2 nt overhang is at the 3′-end of the antisense. Optionally, the dsRNAfurther comprises a ligand (preferably GalNAc₃).

In one embodiment, the dsRNA agent of the invention comprises a senseand antisense strands, wherein: the sense strand is 25-30 nucleotideresidues in length, wherein starting from the 5′ terminal nucleotide(position 1) positions 1 to 23 of said first strand comprise at least 8ribonucleotides; antisense strand is 36-66 nucleotide residues in lengthand, starting from the 3′ terminal nucleotide, comprises at least 8ribonucleotides in the positions paired with positions 1-23 of sensestrand to form a duplex; wherein at least the 3′ terminal nucleotide ofantisense strand is unpaired with sense strand, and up to 6 consecutive3′ terminal nucleotides are unpaired with sense strand, thereby forminga 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′terminus of antisense strand comprises from 10-30 consecutivenucleotides which are unpaired with sense strand, thereby forming a10-30 nucleotide single stranded 5′ overhang; wherein at least the sensestrand 5′ terminal and 3′ terminal nucleotides are base paired withnucleotides of antisense strand when sense and antisense strands arealigned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when said double stranded nucleic acid is introduced into amammalian cell; and wherein the sense strand contains at least one motifof three 2′-F modifications on three consecutive nucleotides, where atleast one of the motifs occurs at or near the cleavage site. Theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at or near the cleavagesite.

In one embodiment, the dsRNA agent of the invention comprises a senseand antisense strands, wherein said dsRNA agent comprises a first strandhaving a length which is at least 25 and at most 29 nucleotides and asecond strand having a length which is at most 30 nucleotides with atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at position 11, 12, 13 from the 5′ end; wherein said 3′ endof said first strand and said 5′ end of said second strand form a bluntend and said second strand is 1-4 nucleotides longer at its 3′ end thanthe first strand, wherein the duplex region region which is at least 25nucleotides in length, and said second strand is sufficientlycomplemenatary to a target mRNA along at least 19 nt of said secondstrand length to reduce target gene expression when said dsRNA agent isintroduced into a mammalian cell, and wherein dicer cleavage of saiddsRNA preferentially results in an siRNA comprising said 3′ end of saidsecond strand, thereby reducing expression of the target gene in themammal. Optionally, the dsRNA agent further comprises a ligand.

In one embodiment, the sense strand of the dsRNA agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the dsRNA agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For dsRNA agent having a duplex region of 17-23 nt in length, thecleavage site of the antisense strand is typically around the 10, 11 and12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe dsRNA from the 5′-end.

The dsRNA agent of the invention may further comprise at least onephosphorothioate or methylphosphonate internucleotide linkage. Thephosphorothioate or methylphosphonate internucleotide linkagemodification may occur on any nucleotide of the sense strand orantisense strand or both in any position of the strand. For instance,the internucleotide linkage modification may occur on every nucleotideon the sense strand or antisense strand; each internucleotide linkagemodification may occur in an alternating pattern on the sense strand orantisense strand; or the sense strand or antisense strand may containboth internucleotide linkage modifications in an alternating pattern.The alternating pattern of the internucleotide linkage modification onthe sense strand may be the same or different from the antisense strand,and the alternating pattern of the internucleotide linkage modificationon the sense strand may have a shift relative to the alternating patternof the internucleotide linkage modification on the antisense strand.

In one embodiment, the dsRNA comprises the phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within duplex region. For example, at least 2, 3, 4, or allthe overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paried nucleotide next to the overhangnucleotide. Preferably, these terminal three nucleotides may be at the3′-end of the antisense strand.

In one embodiment, the dsRNA agent of the invention comprisesmismatch(es) with the target, within the duplex, or combinationsthereof. The mistmatch can occur in the overhang region or the duplexregion. The base pair can be ranked on the basis of their propensity topromote dissociation or melting (e.g., on the free energy of associationor dissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:A:U is preferred over G:C; G:U is preferred over G:C; and I:C ispreferred over G:C (I=inosine). Mismatches, e.g., non-canonical or otherthan canonical pairings (as described elsewhere herein) are preferredover canonical (A:T, A:U, G:C) pairings; and pairings which include auniversal base are preferred over canonical pairings.

In one embodiment, the dsRNA agent of the invention comprises at leastone of the first 1, 2, 3, 4, or 5 base pairs within the duplex regionsfrom the 5′-end of the antisense strand can be chosen independently fromthe group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonicalor other than canonical pairings or pairings which include a universalbase, to promote the dissociation of the antisense strand at the 5′-endof the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In one embodiment, the sense strand sequence may be represented byformula (I):

5′n _(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n _(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification;

wherein at least one of n, N, X, Y, or Z is a xylo modified of formula(A) or a 3′-modified of formula (B); and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the dsRNA agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas, wherein at least one of n, N, X, Y, or Z is a xylomodified of formula (A) or a 3′-modified of formula (B):

5′n _(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Ia);

5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′  (Ib); or

5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Ic).

When the sense strand is represented by formula (Ia), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In one embodiment, the antisense strand sequence of the dsRNA may berepresented by formula (II):

5′n _(q)-N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(X′X′X′)_(l)—N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

wherein at least one of n, N, X, Y, or Z is a xylo modified of formula(A) or a 3′-modified of formula (B)

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the dsRNA agent has a duplex region of 17-23nt in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11,12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, withthe count starting from the 1^(st) nucleotide, from the 5′-end; oroptionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occursat positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas, wherein at least one of n, N, X, Y, or Z is a xylo modified offormula (A) or a 3′-modified of formula (B):

5′n _(q)-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(a)′-n _(p)3′  (IIa);

5′n _(q)-N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n _(p)3′  (IIb); or

5′n _(q)-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(b)′—X′X′X′—N_(a)′-n_(p)3′  (IIc).

When the antisense strand is represented by formula (IIa), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand furthercomprise LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl,2′-C-allyl, or 2′-fluoro. For example, each nucleotide of the sensestrand and antisense strand is independently modified with 2′-O-methylor 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may representa 2′-O-methyl modification or a 2′-fluoro modification, and at least oneof n, N, X, Y, or Z is a xylo modified of formula (A) or a 3′-modifiedof formula (B).

In one embodiment, the sense strand of the dsRNA agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification, and at least one of n, N, X, Y, or Zis a xylo modified of formula (A) or a 3′-modified of formula (B).

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification, and at least one of n, N, X, Y, or Z is a xylo modified offormula (A).

The sense strand represented by any one of the above formulas (Ia), (Ib)and (Ic) forms a duplex with a antisense strand being represented by anyone of formulas (IIa), (IIb) and (IIc), respectively.

Accordingly, the dsRNA agent may comprise a sense strand and anantisense strand, each strand having 14 to 30 nucleotides, the dsRNAduplex represented by formula (III):

sense: 5′n _(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

-   -   p and q are each independently 0-6;    -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 modified nucleotides,        each sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein at least one of n, N, X, Y, or Z is a xylo modified of formula(A)

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q) independently represents anoverhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both iand j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is1; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga dsRNA duplex include the formulas below, wherein at least one of n, N,X, Y, or Z is a xylo modified of formula (I):

5′n _(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′

3′n _(p)′-N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a) ′n _(q)′5′  (IIIa)

5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′

3′n _(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n _(q)5′  (IIIb)

5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′

3′n _(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)′-n_(q)′5′  (IIIc)

When the dsRNA agent is represented by formula (IIIa), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the dsRNA agent is represented as formula (IIIb), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. EachN_(a) independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNA agent is represented as formula (IIIc), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. EachN_(a), N_(a)′ independently represents an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a),N_(a)′, N_(b) and N_(b)′ independently comprises modifications ofalternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and (IIIc) may bethe same or different from each other.

When the dsRNA agent is represented by formula (III), (IIIa), (IIIb) or(IIIc), at least one of the Y nucleotides may form a base pair with oneof the Y′ nucleotides. Alternatively, at least two of the Y nucleotidesform base pairs with the corresponding Y′ nucleotides; or all three ofthe Y nucleotides all form base pairs with the corresponding Y′nucleotides.

When the dsRNA agent is represented by formula (IIIa) or (IIIc), atleast one of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNA agent is represented as formula (IIIb) or (IIIc), atleast one of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, the dsRNA agent of the invention is a multimercontaining at least two duplexes represented by formula (III), (IIIa),(IIIb) or (IIIc), wherein said duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, said multimerfurther comprise a ligand. Each of the dsRNA can target the same gene ortwo different genes; or each of the dsRNA can target same gene at twodifferent target sites.

In one embodiment, the dsRNA agent of the invention is a multimercontaining three, four, five, six or more duplexes represented byformula (III), (IIIa), (IIIb) or (IIIc), wherein said duplexes areconnected by a linker. The linker can be cleavable or non-cleavable.Optionally, said multimer further comprises a ligand. Each of the dsRNAcan target the same gene or two different genes; or each of the dsRNAcan target same gene at two different target sites.

In one embodiment, two dsRNA agent represented by formula (III), (IIIa),(IIIb) or (IIIc) are linked to each other at the 5′ end, and one or bothof the 3′ ends of the are optionally conjugated to to a ligand. Each ofthe dsRNA can target the same gene or two different genes; or each ofthe dsRNA can target same gene at two different target sites.

Various publications described multimeric siRNA and can all be used withthe dsRNA of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 which are hereby incorporated by their entirely.

The dsRNA agent that contains conjugations of one or more carbohydratemoieties to a dsRNA agent can optimize one or more properties of thedsRNA agent. In many cases, the carbohydrate moiety will be attached toa modified subunit of the dsRNA agent. E.g., the ribose sugar of one ormore ribonucleotide subunits of a dsRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier towhich is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

In embodiment the dsRNA of the invention is conjugated to a ligand via acarrier, wherein the carrier can be cyclic group or acyclic group;preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and and decalin; preferably, the acyclic group isselected from serinol backbone or diethanolamine backbone.

The double-stranded RNA (dsRNA) agent of the invention may optionally beconjugated to one or more ligands. The ligand can be attached to thesense strand, antisense strand or both strands, at the 3′-end, 5′-end orboth ends. For instance, the ligand may be conjugated to the sensestrand, in particular, the 3′-end of the sense strand.

Ligands

A wide variety of entities can be coupled to the oligonucleotides of thepresent invention. Preferred moieties are ligands, which are coupled,preferably covalently, either directly or indirectly via an interveningtether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, receptor e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand. Ligands providingenhanced affinity for a selected target are also termed targetingligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In one embodiment, the endosomolytic ligand assumes itsactive conformation at endosomal pH. The “active” conformation is thatconformation in which the endosomolytic ligand promotes lysis of theendosome and/or transport of the composition of the invention, or itscomponents, from the endosome to the cytoplasm of the cell. Exemplaryendosomolytic ligands include the GALA peptide (Subbarao et al.,Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J.Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, theendosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched.

Ligands can improve transport, hybridization, and specificity propertiesand may also improve nuclease resistance of the resultant natural ormodified oligoribonucleotide, or a polymeric molecule comprising anycombination of monomers described herein and/or natural or modifiedribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 2 shows some examples of targetingligands and their associated receptors.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases or a chelator(e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the oligonucleotide into the cellby activating an inflammatory response, for example. Exemplary ligandsthat would have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, naproxen or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. In another alternative, the peptide moiety caninclude a hydrophobic membrane translocation sequence (MTS). Anexemplary hydrophobic MTS-containing peptide is RFGF having the aminoacid sequence AAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP) containing a hydrophobic MTS can also be atargeting moiety. The peptide moiety can be a “delivery” peptide, whichcan carry large polar molecules including peptides, oligonucleotides,and protein across cell membranes. For example, sequences from the HIVTat protein (GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein(RQIKIWFQNRRMKWKK) have been found to be capable of functioning asdelivery peptides. A peptide or peptidomimetic can be encoded by arandom sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide orpeptidomimetic tethered to an iRNA agent via an incorporated monomerunit is a cell targeting peptide such as an arginine-glycine-asparticacid (RGD)-peptide, or RGD mimic. A peptide moiety can range in lengthfrom about 5 amino acids to about 40 amino acids. The peptide moietiescan have a structural modification, such as to increase stability ordirect conformational properties. Any of the structural modificationsdescribed below can be utilized. An RGD peptide moiety can be used totarget a tumor cell, such as an endothelial tumor cell or a breastcancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). AnRGD peptide can facilitate targeting of an iRNA agent to tumors of avariety of other tissues, including the lung, kidney, spleen, or liver(Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGDpeptide will facilitate targeting of an iRNA agent to the kidney. TheRGD peptide can be linear or cyclic, and can be modified, e.g.,glycosylated or methylated to facilitate targeting to specific tissues.For example, a glycosylated RGD peptide can deliver an iRNA agent to atumor cell expressing α_(V)β₃ (Haubner et al., Jour. Nucl. Med.,42:326-336, 2001). Peptides that target markers enriched inproliferating cells can be used. E.g., RGD containing peptides andpeptidomimetics can target cancer cells, in particular cells thatexhibit an integrin. Thus, one could use RGD peptides, cyclic peptidescontaining RGD, RGD peptides that include D-amino acids, as well assynthetic RGD mimics. In addition to RGD, one can use other moietiesthat target the integrin ligand. Generally, such ligands can be used tocontrol proliferating cells and angiogeneis. Preferred conjugates ofthis type lignads that targets PECAM-1, VEGF, or other cancer gene,e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide can be an amphipathic α-helicalpeptide. Exemplary amphipathic α-helical peptides include, but are notlimited to, cecropins, lycotoxins, paradaxins, buforin, CPF,bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clavapeptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Examplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbaone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S.patent application Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether, e.g. a carrier described herein. The ligand ortethered ligand may be present on a monomer when said monomer isincorporated into the growing strand. In some embodiments, the ligandmay be incorporated via coupling to a “precursor” monomer after said“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporatedinto a growing oligonucelotide strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor monomer bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction may be incorporated e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used,although the ligand is typically a carbohydrate e.g. monosaccharide(such as GalNAc), disaccharide, trisaccharide, tetrasaccharide,polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include thosediscussed above. For example, the ligand can be one or more GalNAc(N-acetylglucosamine) derivatives attached through a bivalent ortrivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to abivalent and trivalent branched linkers include the structures shown inany of formula (IV)-(VII):

wherein:

q^(2A), q^(2B), q^(3A), q^(3B), q^(4A), q^(4B), q^(5A), q^(5B), q^(5C)for each represent independently occurrence 0-20 and wherein therepeating unit can be the same or different;

p^(2A), p^(2B), p^(3A), p^(3B), p^(4A), p^(4B), p^(5A), p^(5B), p^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C),are independently for each occurrence absent, alkylene, substitutedalkylene wherin one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and

R^(a) is H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for usewith RNAi agents for inhibiting the expression of a target gene, such asthose of formula (VII):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thefollowing compounds:

DEFINITIONS

As used herein, the terms “dsRNA”, “siRNA”, “RNAi agent”, and “iRNAagent” are used interchangeably to agents that can mediate silencing ofa target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes aprotein. For convenience, such mRNA is also referred to herein as mRNAto be silenced. Such a gene is also referred to as a target gene. Ingeneral, the RNA to be silenced is an endogenous gene or a pathogengene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs,can also be targeted.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent (e.g., dsRNA or siRNA). If the iRNAagent is a single strand it is particularly preferred that it include a5′ modification which includes one or more phosphate groups or one ormore analogs of a phosphate group.

In one embodiment, iRNA agent can have any architecture. E.g., an iRNAagent can have an overhang structure, a hairpin, or other single strandstructure, or a two-strand structure, as described herein.

iRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencingcomplex)); and, molecules which are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed sRNA agents or shorter iRNA agentsherein. “sRNA agent or shorter iRNA agent (siRNA)” as used herein,refers to an iRNA agent, e.g., a double stranded RNA agent or singlestrand agent, that is sufficiently short that it does not induce adeleterious interferon response in a human cell, e.g., it has a duplexedregion of less than 60 but preferably less than 50, 40, or 30 nucleotidepairs. The sRNA agent, or a cleavage product thereof, can down regulatea target gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

The term “single-stranded RNAi” or “ssRNAi” agent or molecule is an RNAiagent that is a single-stranded, nucleic acid-derived molecule having anucleotide sequence that is partially, substantially, or perfectlycomplementary to a nucleotide sequence in a target nucleic acid moleculeor a portion thereof. A second nucleotide sequence with which thesingle-stranded RNAi agent forms base-pairs is not present. Asingle-stranded RNAi molecule can further comprise a terminal phosphategroup located at one or both of the terminal ends, such as a5′-phosphate or a 5′,3′-diphosphate. An ssRNAi molecule/agent caninclude a miRNA or a miRNA mimetic. A single-stranded RNAi agent of theinvention can be loaded into or otherwise associated with RISC andparticipate in gene silencing via an RNAi mechanism. A single-strandedRNAi molecule of the invention can comprise substitutions,chemically-modified nucleotides, and non-nucleotides. A single-strandedRNAi molecule of the invention can comprise one or more or allribonucleotides. Certain embodiments of the invention includesingle-stranded RNAi molecules that comprise substitutions ormodifications in the backbone, sugars, bases, or nucleosides.

The term “miRNA” or “microRNA” is used herein in accordance with itsordinary meaning in the art and refers to small, non-protein coding RNAmolecules that are expressed in a diverse array of eukaryotes, includingmammals, and are involved in RNA-based gene regulation. Mature, fullyprocessed miRNAs are about 15 to about 30 nucleotides in length. Arepresentative set of known, endogenous miRNA species is described inthe publicly available miRBase sequence database, described inGriffith-Jones et al., Nucleic Acids Research, 2004, 32:D109-D111 andGriffith-Jones et al, Nucleic Acids Research, 2006, 34:D 140-D144, andaccessible on the World Wide Web at the Wellcome Trust Sanger Institutewebsite. The mature, fully-processed miRNAs that are publicly availableon the miRBase sequence database are each incorporated by referenceherein. A representative set of miRNAs is also included herein.

The term “miRNA mimetic,” as used herein, refers to a single-strandedRNA molecule that is a mimetic of a naturally-occurring miRNA in a cell.A miRNA mimetic is typically designed based on a corresponding,endogenous miRNA. A miRNA mimetic is capable of modulating theexpression of a target mRNA that is also regulated by a corresponding,naturally-occurring miRNA. A single-stranded RNAi molecule of thepresent invention that is also a miRNA mimetic can be loaded into orotherwise associated with RISC and participates in gene silencing via anRNAi mechanism. A miRNA mimetic of the invention can comprisesubstitutions, chemically-modified nucleotides, and non-nucleotides. AmiRNA mimetic of the invention can comprise one or more or allribonucleotides. Certain embodiments of the invention include miRNAmimetics that comprise substitutions or modifications in the backbone,sugars, bases, or nucleosides. A naturally-occurring miRNA in a cell isreferred to herein as “the corresponding miRNA,” “the endogenous miRNA,”or the “naturally-occurring miRNA.” A single-stranded miRNA mimetic ofthe invention that is provided to a cell is also understood to targetone or more target mRNAs that are also targeted by a corresponding,naturally-occurring miRNA. It is contemplated that a miRNA mimetic ofthe present invention introduced to a cell is capable of functioning asa naturally-occurring miRNA under appropriate conditions.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an siRNA agent of 21 to 23nucleotides.

As used herein, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, a dsRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA, such that the dsRNA agent silencesproduction of protein encoded by the target mRNA. In another embodiment,the dsRNA agent is “exactly complementary” to a target RNA, e.g., thetarget RNA and the dsRNA duplex agent anneal, for example to form ahybrid made exclusively of Watson-Crick base pairs in the region ofexact complementarity. A “sufficiently complementary” target RNA caninclude an internal region (e.g., of at least 10 nucleotides) that isexactly complementary to a target RNA. Moreover, in some embodiments,the dsRNA agent specifically discriminates a single-nucleotidedifference. In this case, the dsRNA agent only mediates RNAi if exactcomplementary is found in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acidmolecule (RNA or DNA) for example of length less than 100, 200, 300, or400 nucleotides.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine. The term “alkyl” refers to saturated and unsaturatednon-aromatic hydrocarbon chains that may be a straight chain or branchedchain, containing the indicated number of carbon atoms (these includewithout limitation propyl, allyl, or propargyl), which may be optionallyinserted with N, O, or S. For example, C₁-C₁₀ indicates that the groupmay have from 1 to 10 (inclusive) carbon atoms in it. The term “alkoxy”refers to an —O-alkyl radical. The term “alkylene” refers to a divalentalkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent speciesof the structure —O—R—O—, in which R represents an alkylene. The term“aminoalkyl” refers to an alkyl substituted with an aminoThe term“mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an—S-alkyl radical.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include trizolyl, tetrazolyl, piperazinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, amino alkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent can be further substituted.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O) (ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above. As used herein,“carbohydrate” refers to a compound which is either a carbohydrate perse made up of one or more monosaccharide units having at least 6 carbonatoms (which may be linear, branched or cyclic) with an oxygen, nitrogenor sulfur atom bonded to each carbon atom; or a compound having as apart thereof a carbohydrate moiety made up of one or more monosaccharideunits each having at least six carbon atoms (which may be linear,branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded toeach carbon atom. Representative carbohydrates include the sugars(mono-, di-, tri- and oligosaccharides containing from about 4-9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C₅and above (preferably C₅-C₈) sugars; di- and trisaccharides includesugars having two or three monosaccharide units (preferably C₅-C₈).

Alternative Embodiments

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene in vivo. The dsRNA agentcomprises a sense strand and an antisense strand, each strand having 14to 30 nucleotides. The sense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the antisensestrand. Every nucleotide in the sense strand and antisense strand hasbeen modified. The modifications on sense strand and antisense strandeach independently comprises at least two different modifications.

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene in vivo. The dsRNA agentcomprises a sense strand and an antisense strand, each strand having 14to 30 nucleotides. The sense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the antisensestrand. The antisense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides. Themodification pattern of the antisense strand is shifted by one or morenucleotides relative to the modification pattern of the sense strand.

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene in vivo. The dsRNA agentcomprises a sense strand and an antisense strand, each strand having 14to 30 nucleotides. The sense strand contains at least two motifs ofthree identical modifications on three consecutive nucleotides, when atleast one of the motifs occurs at the cleavage site in the strand and atleast one of the motifs occurs at another portion of the strand that isseparated from the motif at the cleavage site by at least onenucleotide. The antisense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the strand andat least one of the motifs occurs at another portion of the strand thatis separated from the motif at or near cleavage site by at least onenucleotide.

In another embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene in vivo. The dsRNA agentcomprises a sense strand and an antisense strand, each strand having 14to 30 nucleotides. The sense strand contains at least two motifs ofthree identical modifications on three consecutive nucleotides, where atleast one of the motifs occurs at the cleavage site in the strand and atleast one of the motifs occurs at another portion of the strand that isseparated from the motif at the cleavage site by at least onenucleotide. The antisense strand contains at least one motif of threeidentical modifications on three consecutive nucleotides, where at leastone of the motifs occurs at or near the cleavage site in the strand andat least one of the motifs occurs at another portion of the strand thatis separated from the motif at or near cleavage site by at least onenucleotide. The modification in the motif occurring at the cleavage sitein the sense strand is different than the modification in the motifoccurring at or near the cleavage site in the antisense strand. Inanother embodiment, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene in vivo. The dsRNA agentcomprises a sense strand and an antisense strand, each strand having 12to 30 nucleotides. The sense strand contains at least one motif of three2′-F modifications on three consecutive nucleotides, where at least oneof the motifs occurs at the cleavage site in the strand. The antisensestrand contains at least one motif of three 2′-O-methyl modifications onthree consecutive nucleotides.

The sense strand may further comprises one or more motifs of threeidentical modifications on three consecutive nucleotides, where the oneor more additional motifs occur at another portion of the strand that isseparated from the three 2′-F modifications at the cleavage site by atleast one nucleotide. The antisense strand may further comprises one ormore motifs of three identical modifications on three consecutivenucleotides, where the one or more additional motifs occur at anotherportion of the strand that is separated from the three 2′-O-methylmodifications by at least one nucleotide. At least one of thenucleotides having a 2′-F modification may form a base pair with one ofthe nucleotides having a 2′-O-methyl modification.

In one embodiment, the dsRNA of the invention is administered in buffer.

In one embodiment, siRNA compounds described herein can be formulatedfor administration to a subject. A formulated siRNA composition canassume a variety of states. In some examples, the composition is atleast partially crystalline, uniformly crystalline, and/or anhydrous(e.g., less than 80, 50, 30, 20, or 10% water). In another example, thesiRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the siRNAcomposition is formulated in a manner that is compatible with theintended method of administration, as described herein. For example, inparticular embodiments the composition is prepared by at least one ofthe following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A siRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a siRNA,e.g., a protein that complexes with siRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the siRNA preparation includes another siNA compound,e.g., a second siRNA that can mediate RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent siRNA species. Such siRNAs can mediate RNAi with respect to asimilar number of different genes.

In one embodiment, the siRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than a RNA or a DNA). Forexample, a siRNA composition for the treatment of a viral disease, e.g.,HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, a siRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

Exemplary formulations are discussed below.

Liposomes.

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified siRNAcompounds. It may be understood, however, that these formulations,compositions and methods can be practiced with other siRNA compounds,e.g., modified siRNAs, and such practice is within the invention. AnsiRNA compound, e.g., a double-stranded siRNA compound, or ssiRNAcompound, (e.g., a precursor, e.g., a larger siRNA compound which can beprocessed into a ssiRNA compound, or a DNA which encodes an siRNAcompound, e.g., a double-stranded siRNA compound, or ssiRNA compound, orprecursor thereof) preparation can be formulated for delivery in amembranous molecular assembly, e.g., a liposome or a micelle. As usedherein, the term “liposome” refers to a vesicle composed of amphiphiliclipids arranged in at least one bilayer, e.g., one bilayer or aplurality of bilayers. Liposomes include unilamellar and multilamellarvesicles that have a membrane formed from a lipophilic material and anaqueous interior. The aqueous portion contains the siRNA composition.The lipophilic material isolates the aqueous interior from an aqueousexterior, which typically does not include the siRNA composition,although in some examples, it may. Liposomes are useful for the transferand delivery of active ingredients to the site of action. Because theliposomal membrane is structurally similar to biological membranes, whenliposomes are applied to a tissue, the liposomal bilayer fuses withbilayer of the cellular membranes. As the merging of the liposome andcell progresses, the internal aqueous contents that include the siRNAare delivered into the cell where the siRNA can specifically bind to atarget RNA and can mediate RNAi. In some cases the liposomes are alsospecifically targeted, e.g., to direct the siRNA to particular celltypes.

A liposome containing a siRNA can be prepared by a variety of methods.In one example, the lipid component of a liposome is dissolved in adetergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The siRNApreparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the siRNA andcondense around the siRNA to form a liposome. After condensation, thedetergent is removed, e.g., by dialysis, to yield a liposomalpreparation of siRNA.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Further description of methods for producing stable polynucleotidedelivery vehicles, which incorporate a polynucleotide/cationic lipidcomplex as structural components of the delivery vehicle, are describedin, e.g., WO 96/37194. Liposome formation can also include one or moreaspects of exemplary methods described in Felgner, P. L. et al., Proc.Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S.Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson,et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl.Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; andFukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques forpreparing lipid aggregates of appropriate size for use as deliveryvehicles include sonication and freeze-thaw plus extrusion (see, e.g.,Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidizationcan be used when consistently small (50 to 200 nm) and relativelyuniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta775:169, 1984). These methods are readily adapted to packaging siRNApreparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged entrap nucleicacid molecules rather than complex with them. Since both the nucleicacid molecules and the lipid are similarly charged, repulsion ratherthan complex formation occurs. Nevertheless, some nucleic acid moleculesare entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver siRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated siRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxyl)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of siRNA (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer siRNA, into the skin. In some implementations,liposomes are used for delivering siRNA to epidermal cells and also toenhance the penetration of siRNA into dermal tissues, e.g., into skin.For example, the liposomes can be applied topically. Topical delivery ofdrugs formulated as liposomes to the skin has been documented (see,e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino,R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T.et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176,1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527,1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith siRNA are useful for treating a dermatological disorder.

Liposomes that include siRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include siRNA can bedelivered, for example, subcutaneously by infection in order to deliversiRNA to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Surfactants.

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified siRNAcompounds. It may be understood, however, that these formulations,compositions and methods can be practiced with other siRNA compounds,e.g., modified siRNA compounds, and such practice is within the scope ofthe invention. Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes (see above). siRNA(or a precursor, e.g., a larger dsiRNA which can be processed into asiRNA, or a DNA which encodes a siRNA or precursor) compositions caninclude a surfactant. In one embodiment, the siRNA is formulated as anemulsion that includes a surfactant. The most common way of classifyingand ranking the properties of the many different types of surfactants,both natural and synthetic, is by the use of the hydrophile/lipophilebalance (HLB). The nature of the hydrophilic group provides the mostuseful means for categorizing the different surfactants used informulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker,Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical products and are usable over a wide range of pH values.In general their HLB values range from 2 to about 18 depending on theirstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters.Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and Other Membranous Formulations.

For ease of exposition the micelles and other formulations, compositionsand methods in this section are discussed largely with regard tounmodified siRNA compounds. It may be understood, however, that thesemicelles and other formulations, compositions and methods can bepracticed with other siRNA compounds, e.g., modified siRNA compounds,and such practice is within the invention. The siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor,e.g., a larger siRNA compound which can be processed into a ssiRNAcompound, or a DNA which encodes an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, or precursorthereof)) composition can be provided as a micellar formulation.“Micelles” are defined herein as a particular type of molecular assemblyin which amphipathic molecules are arranged in a spherical structuresuch that all the hydrophobic portions of the molecules are directedinward, leaving the hydrophilic portions in contact with the surroundingaqueous phase. The converse arrangement exists if the environment ishydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

Particles.

For ease of exposition the particles, formulations, compositions andmethods in this section are discussed largely with regard to modifiedsiRNA compounds. It may be understood, however, that these particles,formulations, compositions and methods can be practiced with other siRNAcompounds, e.g., unmodified siRNA compounds, and such practice is withinthe invention. In another embodiment, an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor,e.g., a larger siRNA compound which can be processed into a ssiRNAcompound, or a DNA which encodes an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, or precursorthereof) preparations may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

Pharmaceutical Compositions

The iRNA agents of the invention may be formulated for pharmaceuticaluse. Pharmaceutically acceptable compositions comprise atherapeutically-effective amount of one or more of the the dsRNA agentsin any of the preceding embodiments, taken alone or formulated togetherwith one or more pharmaceutically acceptable carriers (additives),excipient and/or diluents.

The pharmaceutical compositions may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally. Delivery using subcutaneous or intravenous methods can beparticularly advantageous.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe invention which is effective for producing some desired therapeuticeffect in at least a sub-population of cells in an animal at areasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium state, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; and (22) other non-toxic compatible substancesemployed in pharmaceutical formulations.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 0.1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

iRNA agent preparation can be formulated in combination with anotheragent, e.g., another therapeutic agent or an agent that stabilizes aiRNA, e.g., a protein that complexes with iRNA to form an iRNP. Stillother agents include chelators, e.g., EDTA (e.g., to remove divalentcations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broadspecificity RNAse inhibitor such as RNAsin) and so forth.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure. The patient receiving this treatment is any animal in need,including primates, in particular humans, and other mammals such asequines, cattle, swine and sheep; and poultry and pets in general.

Double-stranded RNAi agents are produced in a cell in vivo, e.g., fromexogenous DNA templates that are delivered into the cell. For example,the DNA templates can be inserted into vectors and used as gene therapyvectors. Gene therapy vectors can be delivered to a subject by, forexample, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of a dsRNA agent and one that produces atranscript that includes the bottom strand of a dsRNA agent. When thetemplates are transcribed, the dsRNA agent is produced, and processedinto siRNA agent fragments that mediate gene silencing.

Routes of Delivery

A composition that includes an iRNA can be delivered to a subject by avariety of routes. Exemplary routes include: intravenous, subcutaneous,topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of iRNA and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compositions of the present invention may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. Administration may be topical(including ophthalmic, vaginal, rectal, intranasal, transdermal), oralor parenteral. Parenteral administration includes intravenous drip,subcutaneous, intraperitoneal or intramuscular injection, or intrathecalor intraventricular administration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Dosage

In one aspect, the invention features a method of administering a dsRNAagent, e.g., a siRNA agent, to a subject (e.g., a human subject). Themethod includes administering a unit dose of the dsRNA agent, e.g., asiRNA agent, e.g., double stranded siRNA agent that (a) thedouble-stranded part is 14-30 nucleotides (nt) long, for example, 21-23nt, (b) is complementary to a target RNA (e.g., an endogenous orpathogen target RNA), and, optionally, (c) includes at least one 3′overhang 1-5 nucleotide long. In one embodiment, the unit dose is lessthan 10 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1,0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kgof bodyweight, and less than 200 nmole of RNA agent (e.g., about4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150,75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075,0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with thetarget RNA. The unit dose, for example, can be administered by injection(e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, ora topical application. In some ebmodiments dosages may be less than 10,5, 2, 1, or 0.1 mg/kg of body weight.

In some embodiments, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject has aviral infection and the modality is an antiviral agent other than adsRNA agent, e.g., other than a siRNA agent. In another embodiment, thesubject has atherosclerosis and the effective dose of a dsRNA agent,e.g., a siRNA agent, is administered in combination with, e.g., aftersurgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a dsRNA agent, e.g., a siRNA agent, (e.g., aprecursor, e.g., a larger dsRNA agent which can be processed into asiRNA agent, or a DNA which encodes a dsRNA agent, e.g., a siRNA agent,or precursor thereof). The maintenance dose or doses can be the same orlower than the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 15 mg/kg of body weight per day, e.g., 10,1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. Themaintenance doses are, for example, administered no more than once every2, 5, 10, or 30 days. Further, the treatment regimen may last for aperiod of time which will vary depending upon the nature of theparticular disease, its severity and the overall condition of thepatient. In certain embodiments the dosage may be delivered no more thanonce per day, e.g., no more than once per 24, 36, 48, or more hours,e.g., no more than once for every 5 or 8 days. Following treatment, thepatient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the composition includes a plurality of dsRNA agentspecies. In another embodiment, the dsRNA agent species has sequencesthat are non-overlapping and non-adjacent to another species withrespect to a naturally occurring target sequence. In another embodiment,the plurality of dsRNA agent species is specific for different naturallyoccurring target genes. In another embodiment, the dsRNA agent is allelespecific.

The inventors have discovered that dsRNA agents described herein can beadministered to mammals, particularly large mammals such as nonhumanprimates or humans in a number of ways.

In one embodiment, the administration of the dsRNA agent, e.g., a siRNAagent, composition is parenteral, e.g., intravenous (e.g., as a bolus oras a diffusible infusion), intradermal, intraperitoneal, intramuscular,intrathecal, intraventricular, intracranial, subcutaneous, transmucosal,buccal, sublingual, endoscopic, rectal, oral, vaginal, topical,pulmonary, intranasal, urethral or ocular. Administration can beprovided by the subject or by another person, e.g., a health careprovider. The medication can be provided in measured doses or in adispenser which delivers a metered dose. Selected modes of delivery arediscussed in more detail below.

The invention provides methods, compositions, and kits, for rectaladministration or delivery of dsRNA agents described herein

Methods of Inhibiting Expression of the Target Gene

Embodiments of the invention also relate to methods for inhibiting theexpression of a target gene. The method comprises the step ofadministering the dsRNA agents in any of the preceding embodiments, inan amount sufficient to inhibit expression of the target gene.

Another aspect the invention relates to a method of modulating theexpression of a target gene in a cell, comprising providing to said cella dsRNA agent of this invention. In one embodiment, the target gene isselected from the group consisting of Factor VII, Eg5, PCSK9, TPX2,apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene,GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepciden,Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene,Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKBgene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene,topoisomerase II alpha gene, mutations in the p73 gene, mutations in thep21 (WAF1/CIP1) gene, mutations in the p27 (KIP1) gene, mutations in thePPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene,mutations in the MIB I gene, mutations in the MTAI gene, mutations inthe M68 gene, mutations in tumor suppressor genes, and mutations in thep53 tumor suppressor gene.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

Examples Example 1 In Vitro Screening of siRNA Duplexes Cell Culture andTransfections:

Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, Va.) were grownto near confluence at 37° C. in an atmosphere of 5% CO₂ in RPMI (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Transfection wascarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl ofsiRNA duplexes per well into a 96-well plate and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotic containing ˜2×10⁴ Hep3B cells were then added to the siRNAmixture. Cells were incubated for either 24 or 120 hours prior to RNApurification. Single dose experiments were performed at 10 nM and 0.1 nMfinal duplex concentration and dose response experiments were done using8, 4 fold serial dilutions with a maximum dose of 10 nM final duplexconcentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl Random primers,0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H₂Oper reaction were added into 5 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)),0.5 μl TTR TaqMan probe (Applied Biosystems cat # HS00174914_m1 (human)cat # Rn00562124_m1 (rat)) and 5 μl Lightcycler 480 probe master mix(Roche Cat #04887301001) per well in a 384 well plate (Roche cat#04887301001). Real time PCR was done in a Roche LC 480 Real Time PCRmachine (Roche). Each duplex was tested in at least two independenttransfections and each transfection was assayed in duplicate, unlessotherwise noted.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 or naïve cells over the same dose range,or to its own lowest dose. IC₅₀s were calculated for each individualtransfection as well as in combination, where a single IC₅₀ was fit tothe data from both transfections.

The results of gene silencing of the exemplary siRNA duplex with variousmotif modifications of the invention are shown in the table below.

Example 2 RNA Synthesis and Duplex Annealing 1. OligonucleotideSynthesis:

All oligonucleotides were synthesized on an AKTAoligopilot synthesizeror an ABI 394 synthesizer. Commercially available controlled pore glasssolid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramiditeswith standard protecting groups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis unless otherwise specified. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcarbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of asequence was achieved by starting the synthesis with the correspondingcarbohydrate solid support. Similarly a cholesterol moiety at the 3′-endwas introduced by starting the synthesis on the cholesterol support. Ingeneral, the ligand moiety was tethered to trans-4-hydroxyprolinol via atether of choice as described in the previous examples to obtain ahydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety wasthen coupled to a solid support via a succinate linker or was convertedto phosphoramidite via standard phosphitylation conditions to obtain thedesired carbohydrate conjugate building blocks. Fluorophore labeledsiRNAs were synthesized from the corresponding phosphoramidite or solidsupport, purchased from Biosearch Technologies. The oleyl lithocholic(GalNAc)₃ polymer support made in house at a loading of 38.6 μmol/gram.The Mannose (Man)₃ polymer support was also made in house at a loadingof 42.0 μmol/gram.

Conjugation of the ligand of choice at desired position, for example atthe 5′-end of the sequence, was achieved by coupling of thecorresponding phosphoramidite to the growing chain under standardphosphoramidite coupling conditions unless otherwise specified. Anextended 15 min coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate was introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent The cholesterol phosphoramidite was synthesized in house, andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite was 16 minutes.

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 80 mLof a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at55° C. The bottle was cooled briefly on ice and then the ethanolicammonia mixture was filtered into a new 250 ml bottle. The CPG waswashed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume ofthe mixture was then reduced to ˜30 ml by roto-vap. The mixture was thenfrozen on dry ice and dried under vacuum on a speed vac.

3. Deprotection-II (Removal of 2′ TBDMS Group)

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

4. Analysis

The oligonucleotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides werediluted in water to 150 μl and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. siRNA Preparation

For the preparation of siRNA, equimolar amounts of sense and antisensestrand were heated in 1×PBS at 95° C. for 5 min and slowly cooled toroom temperature. Integrity of the duplex was confirmed by HPLCanalysis.

Example 3 Synthesis of 3′-deoxy-3′-fluoro xylo C and U Nucleosides(Xylo-F C and U)

Example 4 Synthesis of Xylo-O-Methyl (Xylo-OMe) C, U and T Nucleosides

Example 5 Synthesis of Xylo-F U Solid Support and Phosphoramidite

Example 6 Synthesis of Xylo-F C^(Bz) Solid Support and Phosphoramidite

Compound 24: To a solution of compound 10 (4 g, 16.33 mmol) in dry DMF(120 ml) was added benzoic anhydride (17.1 mmol) and the mixture wasstirred at room temperature for 24 h. The solvent was evaporated and theresidue was purified by silica gel column chromatography using agradient of 0-10% methanol in dichloromethane to give 4.76 g of pure 24.

¹H NMR (400 MHz, DMSO) δ 11.31 (s, 1H), 8.25 (d, J=7.4, 1H), 8.01 (d,J=7.5, 2H), 7.63 (t, J=7.4, 1H), 7.52 (t, J=7.7, 2H), 7.36 (d, J=7.3,1H), 5.83 (d, J=6.2, 1H), 5.68-5.52 (m, 2H), 4.34 (dt, J=53.0, 8.7, 1H),3.89 (dt, J=11.1, 6.1, 2H), 3.83-3.71 (m, 1H), 3.30 (d, J=11.0, 1H). ¹⁹FNMR (376 MHz, DMSO) δ −194.52 (m), −194.65 (m). MS: Calcd: 349.11.Found: 350 (M+1).

Compound 25: To a solution of 24 (4.7 g, 13.47 mmol) in dry pyridine (60ml) was added DMTr-Cl (5.42 g, 16 mmol) and the mixture was stirred atroom temperature for 18 h. The reaction mixture was diluted withdichloromethane (200 ml) and washed with water (50 ml). Organic layerwas dried over sodium sulfate and evaporated. The residue wasco-evaporated with toluene and purified by silica gel columnchromatography using a gradient of 0-10% methanol in dichloromethane togive 5.5 g of pure 25.

¹H NMR (400 MHz, DMSO) δ 11.33 (s, 1H), 7.66 (d, J=8.2 Hz, 1H), 7.48 (d,J=7.5 Hz, 2H), 7.41-7.23 (m, 10H), 6.91 (d, J=7.8 Hz, 5H), 5.89 (d,J=5.6 Hz, 1H), 5.55 (d, J=8.1 Hz, 1H), 5.30 (d, J=9.3 Hz, 1H), 4.71 (dt,J=52.6, 8.6 Hz, 1H), 3.91-3.77 (m, 1H), 3.83-3.67 (m, 4H), 3.01 (t,J=11.0 Hz, 1H). ¹⁹F NMR (376 MHz, DMSO) δ −190.92 (m), −191.05 (m). MS:Calcd: 651.24. Found: 650.3 (M−1).

Compound 26: To a solution of 25 (2 g, 3.06 mmol) in dichloromethane (15ml) was added 2-cyanoethyl-tetraisopropylphosphoramidite (1 g, 3.3 mmol)and 4, 5-dicyanoimidazole (0.32 g, 2.75 mmol). The mixture was stirredat room temperature for 18 h, diluted with ethyl acetate (100 ml) andwashed with sodium bicarbonate solution (50 ml). Organic layer was driedover sodium sulfate and evaporated. The residue was subjected to columnchromatography to give 2.4 g of pure 26.

³¹P NMR (162 MHz, CD₃CN) δ 156.12 (d, J=21 Hz), 157.04 (d, J=30.78 Hz).¹⁹F NMR (376 MHz, CD₃CN) δ −188.8 (m), −189.2 (m).

Example 7 Synthesis of Xylo-OMe U and T Phosphoramidite and SolidSupport

The phosphoramidite 29 was prepared from compound 13 as shown in theScheme 5.

The phosphoramidite 30 was prepared from compound 14 as shown in theScheme 5.

The solid supports 33 and 34 were synthesized from compounds 27 and 28respectively as shown in the Scheme 5.

Example 8 Synthesis of Xylo-OMe C Phosphoramidite and Solid Support

The modified oligonucleotides were obtained according to standard solidphase oligonucleotides synthesis and deprotection conditions, and werecharacterized by LC-MS analysis.

TABLE 1 Synthesis and MS analysis of chemically modified siRNAs S/Mass amu siRNA AS Sequence 5′-3′ Calc Found I S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGU_(F)C_(F)U_(F)U_(F)AC_(F)dTsdT6628.95 6627.60 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)C_(F)AGAAU_(F)G 5′ 6726.086725.00 II S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUxC_(F)U_(F)U_(F)AC_(F)dTsdT6640.99 6639.30 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)CxAGAAU_(F)G 5′ 6738.12 6737.00III S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUyC_(F)U_(F)U_(F)AC_(F)dTsdT6628.95 6627.80 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)CyAGAAU_(F)G 5′ 6726.07 6725.00IV S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUzC_(F)U_(F)U_(F)AC_(F)dTsdT6640.98 6639.70 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)CzAGAAU_(F)G 5′ 6738.11 6737.3V S 5′ GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUzC_(F)U_(F)U_(F)AC_(F)dTsdT6640.98 6639.70 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)C_(F)AGAAU_(F)G 5′ 6726.086725.00 VI S 5′GGAU_(F)CxAU_(F)C_(F)U_(F)C_(F)AAGUxC_(F)U_(F)U_(F)AC_(F)dTsdT 6653.036551.90 AS dTsdTC_(F)C_(F)U_(F)AGUxAGAGU_(F)U_(F)CxAGAAU_(F)G 5′ 6750.156748.70 VII S 5′GGAU_(F)CyAU_(F)C_(F)U_(F)C_(F)AAGUyC_(F)U_(F)U_(F)AC_(F)dTsdT 6628.956628.01 AS dTsdTC_(F)C_(F)U_(F)AGUyAGAGU_(F)U_(F)CyAGAAU_(F)G 5′ 6726.086725.20 VIII S 5′GGAU_(F)CzAU_(F)C_(F)U_(F)C_(F)AAGUzC_(F)U_(F)U_(F)AC_(F)dTsdT 6653.026652.50 AS dTsdTC_(F)C_(F)U_(F)AGUzAGAGU_(F)U_(F)CzAGAAU_(F)G 5′ 6750.156749.10 IX S 5′GGAU_(F)CyAU_(F)C_(F)U_(F)C_(F)AAGUyC_(F)U_(F)U_(F)AC_(F)dTsdT 6628.956628.01 AS dTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)C_(F)AGAAU_(F)G 5′6726.08 6725.00 X S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGU_(F)C_(F)U_(F)U_(F)AC_(F)dTsdT6628.95 6627.60 AS dTsdTC_(F)C_(F)U_(F)AGUyAGAGU_(F)U_(F)CyAGAAU_(F)G 5′6726.08 6725.20 Nx: Xylo-OMe U/C; Ny: Xylo-F U/C; Nz: 3′-OMe ribo-U/C;N_(F): 2′-FLC-MS of representative Xylo-F containing oligonucleotide is shown inFIG. 1.

TABLE 2 Tm and IC50 of FVII siRNAs containingXylo-Fluoro and Xylo-OMe sugar modifications Tm ° C. Δ Tm siRNA S/ASSequence (±0.5) wrt II IC50 nM I S 5′ GGAUCAUCUCAAGUCUUACdTsdT 71.8−10.4 0.0095 AS dTsdTCCUAGUAGAGUUCAGAAUG 5′ II S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGU_(F)C_(F)U_(F)U_(F)AC_(F)dTsdT82.2 0 0.0074 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)C_(F)AGAAU_(F)G 5′ III S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUxC_(F)U_(F)U_(F)AC_(F)dTsdT 73.4−8.8 0.0076 AS dTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)CxAGAAU_(F)G 5′IV S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUyC_(F)U_(F)U_(F)AC_(F)dTsdT 70.4−11.8 0.1247 AS dTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)CyAGAAU_(F)G 5′V S 5′ GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUzC_(F)U_(F)U_(F)AC_(F)dTsdT75.5 −6.7 0.0083 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)CzAGAAU_(F)G 5′ VI S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGUzC_(F)U_(F)U_(F)AC_(F)dTsdT 76.3−5.9 0.0086 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)C_(F)AGAAU_(F)G 5′ VII S 5′GGAU_(F)CxAU_(F)C_(F)U_(F)C_(F)AAGUxC_(F)U_(F)U_(F)AC_(F)dTsdT 64.5−17.7  0.1822 AS dTsdTC_(F)C_(F)U_(F)AGUxAGAGU_(F)U_(F)CxAGAAU_(F)G 5′VIII S 5′ GGAU_(F)CyAU_(F)C_(F)U_(F)C_(F)AAGUyC_(F)U_(F)U_(F)AC_(F)dTsdT48.6 −33.6 * AS dTsdTC_(F)C_(F)U_(F)AGUyAGAGU_(F)U_(F)CyAGAAU_(F)G 5′ IXS 5′ GGAU_(F)CzAU_(F)C_(F)U_(F)C_(F)AAGUzC_(F)U_(F)U_(F)AC_(F)dTsdT 70.5−11.7 0.0246 AS dTsdTC_(F)C_(F)U_(F)AGUzAGAGU_(F)U_(F)CzAGAAU_(F)G 5′ XS 5′ GGAU_(F)CyAU_(F)C_(F)U_(F)C_(F)AAGUyC_(F)U_(F)U_(F)AC_(F)dTsdT 61.520.7 0.0100 ASdTsdTC_(F)C_(F)U_(F)AGU_(F)AGAGU_(F)U_(F)C_(F)AGAAU_(F)G 5′ XI S 5′GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGU_(F)C_(F)U_(F)U_(F)AC_(F)dTsdT62.5 19.7 * AS dTsdTC_(F)C_(F)U_(F)AGUyAGAGU_(F)U_(F)CyAGAAU_(F)G 5′Comparison of IC50 values of Xylo-F. Xylo-OMe and 3′-OMe sugar modifiedsiRNAs to the control siRNA is shown in FIG. 2.

Example 8 In Vitro Evaluation of Modified siRNAs

Cells that will be transfected in step 1 should be split or fed one dayprior to beginning the protocol. For each cell line, the number of cellsneeded for transfection, to reach 70-90% confluence 24 hours aftertransfection, should be determined prior to the start of the experiment.

Step 1—Reverse Transfection

-   1.1 For a 10 nM single dose screen, dilute each siRNA to 200 nM in    1×PBS so that 5 μl can be transfected into 100 μl of cells to give a    final siRNA concentration of 10 nM in the well. Serially dilute 1:6    for a total of eight concentrations.-   1.2 To each well of a 96 well plate, add 5 μl of the 200 nM siRNA    solution.-   1.3 Remove cells from the incubator, aspirate media and rinse with    0.25% trypsin to remove any remaining media, which may inactivate    the trypsin.-   1.4 For a 75 cm² culture flask, release cells by adding 3 ml of    0.25% trypsin and incubate at 37° C. until cells float, about 5    minutes.-   1.5 To inactivate the trypsin, add 27 ml of complete media without    antibiotics, as appropriate for the cell type.-   1.6 Pipette cells into a 50 ml conical tube and centrifuge for 3 min    at 1200 RPM.-   1.7 Remove media from cells and replace with 5-10 ml of complete    media without antibiotics.-   1.8 Count cells to determine the total volume of cells that will be    needed for the 96-well plate. At a density of 2×10⁴ cells per well,    approximately 2×10⁶ cells will be needed per plate.-   1.9 Make the cell suspension by resuspending the cells in complete    media such that 80 μl of media contain 2×10⁴ cells.-   1.10 In a separate reservoir, mix 0.2 μl Lipofectamine RNAiMAX with    14.8 μl of Opti-MEM for each well that will be transfected plus an    appropriate amount of overage to account for dead volume (˜10%).    (For each 96-well plate that comes to 22 μl of RNAiMAX and 1.56 mls    of Opti-MEM.)-   1.11 Immediately distribute to the 96 well plates containing the    siRNA and incubate for 20 min. at room temperature.-   1.12 To each well, add 80 μl of the cell suspension.-   1.13 Incubate for 24 hours at the appropriate temperature and CO₂    concentration for the cell line being used.

Step 2. RNA Isolation Using MagMax Magnetic Bead Purification.

-   2.1 Prepare the solutions below for each 96 well plate of RNA to be    purified:    -   Add 6 ml isopropanol to Wash Solution 1.    -   Add 44 ml ethanol to Wash Solution 2.    -   Add 6 ml isopropanol to RNA Rebinding Concentrate.    -   Add 9 ml isopropanol to Lysis Binding Solution Concentrate.    -   Add 110 μl of Turbo DNase to 5.4 ml of MagMax Turbo DNase Buffer        and store on ice until use.-   2.2 Vortex beads for 15 sec. In a sterile reservoir mix 1.1 ml of    beads and 1.1 ml of Lysis/Binding Enhancer for each 96 well plate of    cells.-   2.3 Distribute 20 μl of the bead mixture to each well of a round    bottom plate.-   2.4 Remove cell culture media from cells that were transfected in    step 1.-   2.5 Add 140 μl Lysis/Binding solution to the cells and shake for 1    min at 650 RPM in an eppendorf plate shaker.-   2.6 Add the 140 μl of cell lysate to the beads in the round bottom    plate and shake for 5 minutes at 650 RPM to capture the RNA. Place    the round bottom plate on a magnetic ring stand for 1 minute.-   2.7 With the plate on the magnetic ring stand remove the lysis    mixture using a 12 channel micropipette or by gently inverting the    plate.-   2.8 Add 150 μl Wash Solution 1 and shake for 1 min at 650 RPM. Place    on a magnetic stand for 1 min. then remove the wash solution.-   2.9 Add 150 μl Wash Solution 2 and shake for 1 min at 650 RPM. Place    on a magnetic stand for 1 min then remove the wash solution.-   2.10 Add 50 μl DNase mix to each well and shake at 650 RPM for 15    minutes.-   2.11 Add 100 μl RNA Rebinding Solution to each well and shake for 3    min at 650 RPM to recapture the RNA. Place on a magnetic ring stand    for 1 min and remove Rebinding Solution.-   2.12 Add 150 μl of Wash solution 2 and shake for 1 min at 650 RPM.    Place on a magnetic ring stand for 1 min and then remove wash    solution.-   2.13 To make sure as much liquid is removed as possible before    drying, place the plate on the shaker for 10 seconds at 650 RPM to    gather any remaining liquid at the bottom of the well. Place the    plate on a magnetic ring stand and remove the remaining liquid with    a 12 channel micropipette.-   2.14 Dry the beads by shaking at 650 RPM for 2-5 min.-   2.15 Add 50 μl of RNase free water to the dried beads and shake for    3 min to elute the RNA.-   2.16 Place the plate on a magnetic stand for 1 min then carefully    aspirate 45 μl of eluted RNA without disturbing the magnetic beads.    Place the eluted RNA in a new plate.-   2.17 Measure the concentration of RNA from a subset of wells to    ensure that recover was adequate and consistent across the plate    using a spectrophotometer or Nanodrop    Step 3. cDNA Synthesis

Overview

-   -   In this step cDNA will be generated from the RNA that was        isolated in the step 2. The cDNA will serve as the template for        the qPCR in step 4.

-   3.1 For each RNA sample make a master mix containing 1 μl of 10×    buffer, 0.4 μl of 25× dNTPs, 1 μl of random primers, 0.5 μl of    Reverse transcriptase and 0.5 μl of RNase inhibitor and 1.6 μl of    H₂O. (For each 96-well plate that comes to 100 μl of 10× buffer, 40    μl of 25× dNTPs, 100 μl of random primers, 50 μl of Reverse    transcriptase, 50 μl of RNase inhibitor, and 160 μl of H₂O.)

-   3.2 Distribute 5 μl of master mix to each well of a 96 or 384 well    plate.

-   3.3 Add 5 μl of total RNA isolated in step 2 to each well,    maintaining the plate map that has been established during    transfection.

-   3.4 Centrifuge for 30 sec at 2,000 RPM in a centrifuge equipped to    spin 96 well plates.

-   3.5 Cycle through the following steps using a thermocycler: 25° C.    for 10 min, 37° C. for 120 min, 85° C. for 5 min, and hold at 4° C.    Step 4 qPCR

-   4.1 Make a qPCR master mix containing 0.5 μl of 20× gene specific    TaqMan probe, 0.5 μl of 20× endogenous control TaqMan probe with 5    μl of Roche qPCR master mix and 3 μl of water for each qPCR reaction    plus an appropriate amount of overage to account for dead volume.    (for a 96-well plate that comes to 53 μl of 20× gene specific TaqMan    probe, 53 μl of 20× endogenous control TaqMan probe with 530 μl of    Roche qPCR master mix and 330 μl of water.)

-   4.2 Distribute 8 μl of master mix to 96 or 384 well qPCR plates and    centrifuge at 2,000 RPM for 15 sec to pool master mix at the bottom    of the well.

-   4.3 Add 2 μl of cDNA to the master mix. Replicate qPCR reactions can    be run on the same plate, or on separate plates. Seal plates and    centrifuge at 2,000 RPM for 15 seconds.

-   4.4 Run the qPCR reaction using a method that can detect    fluorescence from both VIC and FAM. On a Roche LightCycler 480 we    use the standard-Dual Color Hydrolysis Probe/UPL Probe program

Example 9 In Vivo Activity of Xylo-Sugar Modified siRNA Duplex

Compound was administered to female C57BL/6 mice (6-10 weeks, 5 pergroup) via subcutaneous injection at a dose volume of 10 μl/g at a doseof 25, and 5 mg/kg siRNA. Control animals received PBS by subcutaneousinjection at the same dose volume.

After approximately 48 hours, mice were anesthetized with 200 μl ofketamine, and then exsanguinated by severing the right caudal artery.Liver tissue was collected, flash-frozen and stored at −80° C. untilprocessing.

Efficacy of treatment was evaluated by measurement of TTR mRNA in liverat 48 hours post-dose. TTR liver mRNA levels were assayed utilizing theBranched DNA assays-QuantiGene 1.0 (Panomics). Briefly, mouse liversamples were ground and tissue lysates were prepared. Liver lysismixture (a mixture of 1 volume of lysis mixture, 2 volume ofnuclease-free water and 10 μl of Proteinase-K/ml for a finalconcentration of 20 mg/ml) was incubated at 65° C. for 35 minutes. 5 ulof liver lysate and 95 μl of working probe set (TTR probe for genetarget and GAPDH for endogenous control) were added into the CapturePlate. Capture Plates were incubated at 53° C.±1° C. (approximately16-20 hours). The next day, the Capture Plates were washed 3 times with1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash BufferComponent 2), then dried by centrifuging for 1 minute at 240 g. 100 μlof Amplifier Probe mix per well was added into the Capture Plate, whichwas sealed with aluminum foil and incubated for 1 hour at 46° C.±1° C.Following 1 hour incubation, the wash step was repeated, then 100 μl ofLabel Probe mix per well was added. Capture plates were incubated at 46°C.±1° C. for 1 hour. The plates were then washed with 1× Wash Buffer,dried and 100 μl Substrate per well was added into the Capture Plates.Capture Plates were incubated for 30 minutes at 46° C. followed byincubation for 30 minutes at room temperature. Plates were read usingthe SpectraMax Luminometer following incubation. bDNA data were analyzedby subtracting the average background from each duplicate sample,averaging the resultant duplicate GAPDH (control probe) and TTR(experimental probe) values, and then computing the ratio: (experimentalprobe-background)/(control probe-background). The average TTR mRNA levelwas calculated for each group and normalized to the PBS group average togive relative TTR mRNA as a percentage of the PBS control group.

Results of the in vivo activity of Xylo-sugar modified siRNA duplexesare shown in FIG. 3. As demonstrated in the figure, Xylo-sugar modifiedsiRNA duplexes were potent in vivo.

Example 10 Stability of Xylo-Sugar Modification Against ExonucleasesSnake Venon Phosphodiesterases (SCPD) Assay

To access the stability of the Xylo-sugar modified siRNA duplex,exonucleases Snake Venon Phosphodiesterases (SCPD) assay was carried outaccording to the protocol described in Rajeev et al. (see Rajeev, K. G.;Prakash, T. P.; Manoharan, M., “2′-Modified-2-thiothymidineOligonucleotides,” Organic Lett. 5 (17): 3005-3008 (2003), and itssupporting information, the content of which are herein incorporated byreference in their entirety).

Results of the stability of the Xylo-sugar modified siRNA againstdexonucleases SCPD assay are shown in FIG. 4.

1. A double-stranded RNAi agent capable of inhibiting the expression ofa target gene, comprising a sense strand and an antisense strand, eachstrand having 12 to 30 nucleotides, wherein the duplex comprises atleast one xylo-modified or 3′-modified modified moiety.
 2. Thedouble-stranded RNAi agent of claim 1, wherein the xylo modified moietyis represented by formula (A):

wherein: Y is H, alkyl or internucleotide linkage; X is halogen,substituted or unsubstituted alkoxy, substituted or unsubstitutedaminoalkyl, substituted or unsubstituted alkyl; and B is a natural ornon-natural nucleobase.
 3. The double-stranded RNAi agent of claim 1,wherein the xylo modified moiety is represented by formula (B):

wherein: Y is H, alkyl or internucleotide linkage; X is halogen,substituted or unsubstituted alkoxy, substituted or unsubstitutedaminoalkyl, substituted or unsubstituted alkyl; and B is a natural ornon-natural nucleobase.
 4. The double-stranded RNAi agent of claim 2,wherein X is F or OMe.
 5. A double-stranded RNAi agent of claim 2,represented by formula (III):sense: 5′n _(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n _(q)3′antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III) wherein: j, k, and l are each independently 0 or 1; p andq are each independently 0-6; each Na and Na′ independently representsan oligonucleotide sequence comprising 0-25 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotideseach Nb and Nb′independently represents an oligonucleotide sequence comprising 0-10nucleotides which are either modified or unmodified or combinationsthereof; each n_(p) and n_(q) independently represents an overhangnucleotide; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ eachindependently represent one motif of three identical modifications onthree consecutive nucleotides; wherein at least one of n, N, X, Y, or Zis a xylo modified of formula (A); and wherein modifications on Nb isdifferent than the modification on Y and modifications on Nb′ isdifferent than the modification on Y′.
 6. The double-stranded RNAi agentof claim 1, wherein the duplex region is 17-30 nucleotide pairs inlength.
 7. The double-stranded RNAi agent of claim 1, wherein the duplexregion is 17-19 nucleotide pairs in length.
 8. The double-stranded RNAiagent of claim 1, wherein the duplex region is 27-30 nucleotide pairs inlength.
 9. The double-stranded RNAi agent of claim 1, wherein eachstrand has 17-30 nucleotides.
 10. The double-stranded RNAi agent ofclaim 1, further comprising at least one ligand.
 11. The double-strandedRNAi agent of claim 10, wherein the ligand is a one or more GalNAcderivatives attached through a bivalent or trivalent branched linker.12. The double-stranded RNAi agent of claim 11, wherein the ligand is


13. The double-stranded RNAi agent of claim 10, wherein the ligand isattached to the 3′ end of the sense strand.
 14. The double-stranded RNAiagent of claim 1, further comprising at least one phosphorothioate ormethylphosphonate internucleotide linkage.
 15. The double-stranded RNAiagent of claim 1, wherein the nucleotide at the 1 position of the 5′-endof the duplex in the antisense strand is selected from the groupconsisting of A, dA, dU, U, and dT.
 16. The double-stranded RNAi agentof claim 1, wherein the base pair at the 1 position of the 5′-end of theduplex is an AU base pair.
 17. The double-stranded RNAi agent of claim5, wherein the Y nucleotides contain a 2′-fluoro modification.
 18. Thedouble-stranded RNAi agent of claim 5 wherein the Y′ nucleotides containa 2′-O-methyl modification.
 19. A pharmaceutical composition comprisingthe double-stranded RNAi agent of claim 1 alone or in combination with apharmaceutically acceptable carrier or excipient.
 20. A method forinhibiting the expression of a target gene comprising the step ofadministering the double-stranded RNAi agent of claim 1, in an amountsufficient to inhibit expression of the target gene.
 21. The method ofclaim 20, wherein the double-stranded RNAi agent is administered throughsubcutaneous or intravenous administration.
 22. The double-stranded RNAiagent of claim 3, wherein X is F or OMe.
 23. The double-stranded RNAiagent of claim 5, further comprising at least one phosphorothioate ormethylphosphonate internucleotide linkage.
 24. The double-stranded RNAiagent of claim 5, wherein the nucleotide at the 1 position of the 5′-endof the duplex in the antisense strand is selected from the groupconsisting of A, dA, dU, U, and dT.
 25. The double-stranded RNAi agentof claim 5, wherein the base pair at the 1 position of the 5′-end of theduplex is an AU base pair.